Method for detecting genes sensitive to low-level ionizing radiation, and gene detected by the method

ABSTRACT

A method for detecting genes sensitive to low-level ionizing radiation and genes detected by the method. More specifically, genes sensitive to low-level ionizing radiation and related to suppressing thymic cancer, discovered in a carcinogenic entity and verified in a normal entity are detected by subjecting a cancerous AKR/J mouse and a normal ICR mouse to low-level radiation. Thymus is collected therefrom, immunogenic and apoptotic genes are classified via microarray processing of the thymus. The genes are amplified and the levels of gene expression are measured. Thus, a gene having a specific reaction to radiation can be accurately detected by preventing the interference of confounding variables.

RELATED APPLICATIONS

This application is a § 371 application from PCT/KR2012/003906 filed May18, 2012, which claims priority from Korean Patent Application No.10-2012-0049560 filed May 10, 2012, each of which is herein incorporatedby reference in its entirety.

REFERENCE TO ELECTRONIC SEQUENCE

The contents of the electronic sequence listing (seq.txt; Size: 2.11kilobytes; and Date of Creation: Nov. 9, 2014) is herein incorporate byreference in its entirety.

TECHNICAL FIELD

The present invention relates to a method for detecting genes sensitiveto a low level of ionizing radiation and genes detected by the method,and more particularly, to a method for detecting genes sensitive to alow level of ionizing radiation, the method including irradiatingcancer-induced mice and normal mice with a low level of radiation, andscreening immunity-related and apoptosis-related genes, which areobserved commonly in the normal mice and the cancer-induced mice, fromthe thymi of the mice.

BACKGROUND ART

With an increase in the industrial and medical use of radiation, variousstudies on the effects of radiation on the human body have beenconducted, and particularly, cancer therapy with radiation has receivedattention. It is known that high doses of ionizing radiation cause DNAdamage, genetic modification, and diseases, including cancer, but aradiation dose of 200 mGy or less and a radiation dose rate of 6 mGy/hror less inhibit cancer development by activating immune responses.

In general, studies on the relationship between radiation and cancerdevelopment, particularly gene responses to radiation, have beenconducted, but confounding factors have significantly affected theresults to reduce the reliability of the results. However, most studiesconducted to date could not explain various responses, which occur inthe cells, tissues and organs of the body in the body stage, becausethese studies were performed using gene-modified cell lines or cancercell lines. In other words, because gene responses were evaluated usinggeneral mice, a variety of genes were expressed, and because cancerdevelopment was not limited to a specific organ, it was difficult toanalyze gene responses.

In prior art methods that use cells for cancer research, genes weremodified, or cancer cells lacking p53 that is important in cancerdevelopment were irradiated. For this reason, there was a problem inthat the results could not be applied to individuals, because they didfundamentally differ from the responses of normal cells. To overcomethis problem, studies on the effects of radiation on cancer developmenthave been conducted using mice having a gene similarity of 95% or morewith humans. However, cancer incidence in general mice is very low, andthus a variety of mouse models for cancer research have been used.

In prior studies, a variety of methods were used to screen genessensitive to a low level (0.7 mGy/hr) of radiation. However, genesdisclosed in the present invention are not yet known as genes sensitiveto a low level (0.7 mGy/hr) of radiation. Technologies prior to theidentification of the profile of genes according to the presentinvention are as follows.

(1) Radiation-sensitive genes were identified in human breast epithelialcells using a gene probe method (Malone J, Ullrich R (2007) Radiat Res167: 176-1847).

(2) Genes sensitive to early radiation were found in irradiated humanlymphocytes using microarray analysis and a quantitative nucleic acidamplification technique (Turtoi A et. al. (2008) Int J Radiat Biol 84:375-387).

(3) Biological dose markers were identified in male BALB/c, irradiatedwith 1-7 Gy, by blood protein analysis (Ossetrova N I, Blakely W F(2009) Int J Radiat Biol 85: 837-850).

(5) A network of 10 herb genes including c-Jun, HDAC1, RELA (p65 subunitof NFKB), PKC-beta, SUMO-1, c-Abl, STAT1, AR, CDK1 and IRF1 wasidentified by linear regression analysis of 500 genes that represent abiological network (Eschrich S et. al. (2009) Int J Radiat Oncol BiolPhys 75: 497-505).

(5) Single nucleotide polymorphism markers enabling the prediction ofresponse to radiation were identified in 277 human lymphocyte cell lines(Niu N et. al. (2010) Genome Res 20: 1482-1492).

(6) A recent study provided a new overview of response to radiationusing EPI-200 that is a three-dimensional tissue model mimicking humanepidermal tissue (Mezentsev A, Amundson S A (2011) Radiat Res 175:677-688).

(7) An RNA-based expression analysis method was used as a tool forbiological dose evaluation in the measurement of radiation exposure andtoxicity (Pogosova-Agadjanyan E L et. al. (2011) Radiat Res 175:172-184).

(8) Radiation-sensitive genes were identified in human lymphocytes andleucocytes using multiplex quantitative nucleic acid amplification and amethod for analysis of a specific gene (Kabacik S et. al. (2011) Int JRadiat Biol 87: 115-129).

Accordingly, the present inventors have made efforts to find genesshowing a sensitive response to a low level of ionizing radiation. As aresult, the present inventors have analyzed the functions of 7 genesplaying an important role in immunity (Ighg, Saa2, Defb6, Reg3g, Tac2and Igh-VJ558) and apoptosis (Klk1b27), and have found that the sevengenes have not been reported, thereby completing the present invention.

DISCLOSURE Technical Problem

It is an object of the present invention to a method for detecting agene sensitive to a low level of ionizing radiation, and a gene detectedby the method.

Technical Solution

In order to accomplish the above object, the present invention providesa method for detecting a gene, which is sensitive to a low level ofradiation, identified in a cancer-induced individual, verified using anormal individual, and involved in suppression of thymic cancer, themethod including the steps of: I) irradiating a cancer-induced AKR/Jmouse and a normal ICR mouse with a low level of radiation; II)extracting thymi from the AKR/J mouse and the ICR mouse; III) analyzingthe thymi by microarray analysis; IV) selecting an immunity-related orapoptosis-related gene from the microarray analysis through Venn diagramanalysis; and V) amplifying the gene and measuring the expression levelof the gene.

The present invention also provides a marker for diagnosing thesuppression of thymic cancer, the marker including either animmunity-related gene selected from the group consisting of Ighg(NM_001472541), Saa2 (NM_011314), Reg3g (NM_011260), Defb6 (NM_054074),Tac2 (NM_009312) and Igh-VJ558 (NM_001474025), which are involved in thesuppression of thymic cancer, or an apoptosis-related gene Klk1b27(NM_020268).

The present invention also provides a kit for diagnosing the suppressionof thymic cancer, the kit including the above marker.

The present invention also provides a microarray for diagnosing thymiccancer, the microarray including the above marker.

The present invention also provides a method for detecting a gene fortreating or suppressing thymic cancer, the method including the stepsof: I) irradiating a mammal having thymic cancer with radiation; II)bringing a test substance into contact with a thymic tissue extractedfrom the irradiated mammal; and III) measuring, from the thymic tissue,a change in the expression of either an immunity-related gene selectedfrom the group consisting of Ighg (NM_001472541), Saa2 (NM_011314),Reg3g (NM_011260), Defb6 (NM_054074), Tac2 (NM_009312) and Igh-VJ558(NM_001474025), which are involved in suppression of thymic cancer, oran apoptosis-related gene Klk1b27 (NM_020268).

Hereinafter, the present invention will be described in detail.

According to the present invention, Ighg and Igh-VJ558, known to beinvolved in immune responses, were activated by a low level ofradiation. It was found that a low level of radiation can activate animmune response during the development of thymic cancer.

Also, it was analyzed that Saa2 is involved in a systemic inflammatoryresponse and the production of free fatty acids (Lopez-Nieva P et. al.(2004) Carcinogenesis 25: 1299-1304). In addition, it was analyzed thatReg3g is involved in an inflammatory response (Beverly L J, Felsher D W(2005) Cancer Res 65: 7159-7168), and that Defb6 is involved in animmune response in genitourinary organs (Yamaguchi Y et. al. (2002) JImmunol 169: 2516-2523).

Moreover, it was analyzed that Tac2 is observed mainly in neuronal cellsand non-neuronal cells and involved in the synthesis of gonadotropin inmales (Corander M P et. al. (2010) J Neuroendocrinol 22:181-187). Inaddition, it was analyzed that Klk1b27 is serine protease and involvedin insulin growth hormone protein binding (REACTOME: 1494374).

Many studies on the effects of radiation on cancer development among theeffects of radiation on the human body have been conducted, but it wasdifficult to explain various responses of the body to radiation(responses of genes to radiation), because these studies were performedusing cancer cells, gene-modified cell lines or general mice.Particularly, the profile of genes sensitive to a low level (0.7 mGy/hr)of radiation in individuals has not yet been identified, and thefunctions of these genes have not been explained. Accordingly, thepresent invention is intended to (1) identify the profile of genes thatare expressed specifically in thymus and sensitive to a low level ofradiation, and analyze the functions of the genes, after irradiating alow level (0.7 mGy/hr) of radiation (cancer development stimulator) tonormal ICR mice and AKR/J mice that develop thymic cancer, and 2)diagnose the stage of development of thymic cancer using the profile ofgenes.

The present invention provides a method for detecting a gene, which isidentified in a cancer-induced individual, is verified using a normalindividual, and is involved in suppression of thymic cancer, the methodincluding the steps of: I) irradiating a cancer-induced AKR/J mouse anda normal ICR mouse with a low level of radiation; II) extracting thymifrom the AKR/J mouse and the ICR mouse; III) analyzing the thymi bymicroarray analysis; IV) selecting an immunity- or apoptosis-relatedgene from the microarray analysis through Venn diagram analysis; and V)amplifying the gene and measuring the expression level of the gene.

In the inventive method for detecting a gene sensitive to a low level ofionizing radiation, irradiating the mice with the low level of radiationis preferably performed by irradiating gamma radiation (Cs-137) at adose rate of 0.7 mGy/hr to a final dose of 1.7 Gy. The method accordingto the present invention is preferably used for preparation of a kit fordiagnosing thymic cancer, evaluation of the degrees of progression andtreatment of cancer in a cancer patient, evaluation of the relationshipbetween radiation exposure of industrial and medical workers and cancerdevelopment, evaluation of the causal relation between radiation andcancer development, biological evaluation of radiation exposure dose, orevaluation of the degrees of development and progression of thymiccancer caused by a low level of radiation.

In addition, the inventive method for detecting a gene sensitive to alow level of ionizing radiation, the cancer is preferably thymic cancer,and extracting the thymi in step II) is preferably performed at a timepoint when the mouse starts to die of the cancer.

Furthermore, in the inventive method for detecting a gene sensitive to alow level of ionizing radiation, the immunity-related gene is preferablyselected from the group consisting of Ighg (NM_001472541), Saa2(NM_011314), Reg3g (NM_011260), Defb6 (NM_054074), Tac2 (NM_009312) andIgh-VJ558 (NM_001474025), and the apoptosis-related gene is preferablyKlk1b27 (NM_020268). Preferably, the Ilghg (NM_001472541) gene isamplified using primers having sequences set forth in SEQ ID NOS: 1 and2; the Saa2 (NM_011314) gene is amplified using primers having sequencesset forth in SEQ ID NOS: 3 and 4; the Reg3g (NM_011260) gene isamplified using primers having sequences set forth in SEQ ID NOS: 5 and6; the Defb6 (NM_054074) gene is amplified using primers havingsequences set forth in SEQ ID NOS: 7 and 8; the Tac2 (NM_009312) gene isamplified using primers having sequences set forth in SEQ ID NOS: 9 and10; the Tac2 (NM_009312) gene is amplified using primers havingsequences set forth in SEQ ID NOS: 11 and 12; and the Klk1b27(NM_020268) gene is amplified using primers having sequences set forthin SEQ ID NOS: 13 and 14.

In step IV) of selecting the related gene from the microarray analysis,a gene overexpressed in the cancer-induced individual after irradiationcompared to in the cancer-induced individual before irradiation isdetected by microarray analysis, and thus verified using primers havingsequences of two of SEQ ID NOS: 1 to 10, and the overexpressed orunderexpressed gene is identified by performing a search for thefunction thereof. The microarray analysis is described in the Examplesbelow, and the overexpressed gene is identified by performing a searchfor the function thereof. The microarray analysis is described in theExamples below, and verification is performed using primers havingsequences of SEQ ID NOS: 1 to 14. A search for the function of the genewas performed in the Examples through BioCarta (www.biocarta.com/),GenMAPP (www.genmapp org/), the DAVID bioinformatics database(apps1.niaid.nih.gov) and the PubMed database (www.ncbi.nlm.nih.gov),but is not limited thereto.

As used herein, “gene sensitive to a low level of radiation” refers to agene that is differentially overexpressed in a cancer-induced individualafter radiation compared to before irradiation. In other words, the generefers to a gene whose expression pattern is changed by stimulation withradiation, and it may be a target gene associated with a specificcancer, that is, an oncogene or a tumor suppressor gene. When thiscancer-specific gene is detected, a molecular mechanism for radiotherapyof cancer patients can be established, which can contribute to anincrease in the effect of radiotherapy, and a platform for thedevelopment of agents or methods for treating cancer at the biomolecularlevel can be provided by screening novel oncogenes or tumor suppressorgenes and regulating the expression thereof.

The present invention also provides a marker for diagnosing thesuppression of thymic cancer, the marker including either animmunity-related gene selected from the group consisting of Ighg(NM_001472541), Saa2 (NM_011314), Reg3g (NM_011260), Defb6 (NM_054074),Tac2 (NM_009312) and Igh-VJ558 (NM_001474025), which are involved in thesuppression of thymic cancer, or an apoptosis-related gene Klk1b27(NM_020268).

The present invention also provides a kit for diagnosing the suppressionof thymic cancer, the kit including the above marker.

The present invention also provides a microarray for diagnosing thymiccancer, the microarray including the above marker.

The present invention also provides a method for detecting a gene fortreating or suppressing thymic cancer, the method including the stepsof: I) irradiating a mammal having thymic cancer with radiation; II)bringing a test substance into contact with a thymic tissue extractedfrom the irradiated mammal; and III) measuring, from the thymic tissue,a change in the expression of either an immunity-related gene selectedfrom the group consisting of Ighg (NM_001472541), Saa2 (NM_011314),Reg3g (NM_011260), Defb6 (NM_054074), Tac2 (NM_009312) and Igh-VJ558(NM_001474025), which are involved in suppression of thymic cancer, oran apoptosis-related gene Klk1b27 (NM_020268).

In the present invention, AKR/J mice (models for thymic cancer research)and healthy ICR mice were irradiated with a low level (0.7 mGy/hr) ofgamma radiation (Cs-137), and thymi were extracted at a time point (day100) when the AKR/J mice started to die of thymic cancer. The extractedthymi were analyzed by microarray analysis, and then immunity- andapoptosis-related genes that responded sensitively to the low level ofradiation (0.7 mGy/hr) were selected through Venn diagram analysis, andsubjected to nucleic acid amplification, and the expression levelsthereof were measured.

As a result, 7 genes, including immunity-related genes (Ighg, Saa2,Defb6, Reg3g, Tac2 and Igh-VJ558) and an apoptosis-related gene(Klk1b27), which responded sensitively to the low level (0.7 mGy/hr) ofradiation, were screened in the present invention. Also, it was foundthat a low level (0.7 mGy/hr) of radiation suppressed thymic cancer byactivating immunity-related genes (Ighg, Saa2, Defb6, Reg3g, Tac2 andIgh-VJ558) and an apoptosis-related gene (Klk1b27) in thymocytes. Asdescribed above, according to the present invention, the functions ofthe related genes were suggested, and the responses of glucosemetabolism-related genes to a low level of radiation could beconsistently observed by extracting thymi at day 100 when death causedby thymic cancer was observed.

Therefore, the present invention may be used to: (1) identify theprofile of genes for development of a kit for diagnosing thymic cancer;(2) identify a marker for evaluating the relation of cause and effect ofcancer development in industrial and medical workers who live inenvironments having a low level of radiation; (3) identify the profileof genes for information, which enable the diagnosis of cancerdevelopment in cancer patients and allow a cancer therapeutic method tobe established; (4) identify a marker for evaluating the causal relationbetween radiation exposure and the development of thymic cancer; 5)identify a novel gene marker that may be widely used for biologicalevaluation of a low level of radiation exposure; and (6) understandionizing radiation-sensitive glucose metabolism signaling that may beused as a target therapy for a low level of radiation exposure.

Advantageous Effects

The method for detecting a gene sensitive to a low level of ionizingradiation as described above may be used to establish the profile ofmarker genes sensitive to a low level of radiation in order to prepare akit for diagnosing thymic cancer, and may provide a marker genesensitive to a low level of radiation, which can be used to evaluate thedegrees of progression and progression of cancer in cancer patients.Also, the method according to the present invention may provide a markergene sensitive to a low level of radiation, which can be used toevaluate the relationship between the radiation exposure of industrialand medical workers and cancer development. Further, it may provide amarker sensitive to a low level of radiation, which can be used toevaluate the causal relation between radiation and cancer development.In addition, it may provide a novel marker that can be used forbiological evaluation of radiation exposure dose. Also, it may provide agene marker sensitive to a low level of radiation (0.7 mGy/hr), whichcan be used to evaluate the low-level radiation on the suppression ofthymic cancer.

DESCRIPTION OF DRAWINGS

FIG. 1 schematically shows that a low level (0.7 mGy/hr) of radiationsuppresses thymic cancer by activating immunity-related genes (Ighg,Saa2, Defb6, Reg3g, Tac2 and Igh-VJ558) and an apoptosis-related gene(Klk1b27) in thymocytes.

FIG. 2 is a graph showing the results obtained by irradiating AKR/J miceICR mice with a low level (0.7 mGy/hr) of radiation and measuring theweight of thymi of the mice at a time point (day 100) when the micestarted to die of thymic cancer during their housing, in order toanalyze the responses of genes sensitive to radiation based on thethymus weight.

MODE FOR INVENTION

Hereinafter, the present invention will be described in further detailwith reference to examples. It is to be understood, however, that theseexamples are for illustrative purposes only and are not intended tolimit the scope of the present invention.

Example 1

6-Week-old female AKR/J mice (models for thymic cancer research) and6-week-old female ICR mice were purchased from SLC Co., Ltd. (Japan). Alow level of radiation (¹³⁷Cs) was irradiated to the AKR/J mice using agamma-ray generator (IBL 147C, CIS bio international, France) at a doserate of 0.7 mGy/hr) so as to reach a final dose of 1.7 Gy. Aftercompletion of irradiation with the low level of radiation, the mice weretransferred into a sterilized housing system shielded from radiation,and were housed therein for 100 days while the observation ofdevelopment of thymic cancer was performed. For gene analysis, under thesame experimental conditions, normal mice (ICR mice) housed separatelyfrom the AKR/J mice were irradiated with a low level of radiation (0.7mGy/hr). After 100 days, thymi were extracted from the mice and frozenrapidly in liquid nitrogen, after which gene analysis was performed.

Example 2: Microarray and Gene Analysis

Using mouse models (AKR/J mice) for cancer research, irradiated inExample 1, genes sensitive to a low level of radiation (0.7 mGy/hr) werescreened. The screened genes were verified using normal mice (ICR mice).Specifically, genes that responded to a low level of radiation (0.7mGy/hr) specifically in the thymi of the AKR/J and ICR mice irradiatedwith the low-level of radiation (0.7 mGy/hr) were screened, and theirfunctions were analyzed. Analysis was performed using Venn diagrams, aquantitative nucleic acid amplification technique, and the statisticalprogram SAS (ANOVA and t-test).

To confirm the results, the genes were subjected to nucleic acidamplification. Specifically, the thymi extracted from the AKR/J and ICRmice irradiated with the low level of radiation (0.7 mGy/hr) weremicroarrayed, and genes that responded sensitively to the low level ofradiation were amplified using the primers shown in Table 1 below inorder to measure the expression levels thereof.

TABLE 1 Gene No. Gene name Forward (5′ -> 3′) Reverse (5′ -> 3′)NM_001472541 Ighg GGGCTAGACCTGGATGATGT GCTGATGAAGATGGTGATGG(SEQ ID NO 1) (SEQ ID NO 2) NM_011314 Saa2 AGCTGGCTGGAAAGATGGAGACAATGTCCTCTGCCGAAGAATTCCTGA (SEQ ID NO 3) (SEQ ID NO 4) NM_011260 Reg3gTCCTGTCCTCCATGATCAAA ACATCAGCATTGCTCCACTC (SEQ ID NO 5) (SEQ ID NO 6)NM_054074 Defb6 TGGTGATGCTGTCTCCACTT CATGAACGCTGGCATGAG (SEQ ID NO 7)(SEQ ID NO 8) NM_009312 Tac2 GGACTTATGGGCAAGAGGAA GGGATTATTTGAGGATGCCA(SEQ ID NO 9) (SEQ ID NO 10) NM_001474025 Igh-VJ558 GAGAAATCCTGAGGGAGCTGAGGACGCTGGACACACTGTA (SEQ ID NO 11) (SEQ ID NO 12) NM_020268 Klk1b27TGTAGGAGAGACGGGTGGA TACCGTGGAGAACACCATCA (SEQ ID NO 13) (SEQ ID NO 14)

After irradiation of the AKR/J and ICR mice with the low level ofradiation (0.7 mGy/hr), the mice were housed, and thymi were extractedfrom the mice at a time point (day 100) when the AKR/J mice started todie of thymic cancer. The extracted thymi were microarrayed, and genesthat responded sensitively to the low level of radiation were selected,and then subjected to nucleic acid amplification, and the expressionlevels thereof were measured. As a result, it was shown that, in themice irradiated with the low level of radiation, immunity-related genes(Ighg, Saa2, Defb6, Reg3g, Tac2 and Igh-VJ558) and an apoptosis-relatedgene (Klk1b27) responded sensitively to the low level of radiation. Theresults are shown in Table 2 below.

TABLE 2 Quantitative nucleic Microarray amplification ICR AKR/J ICRAKR/J Gene No. Gene name mice mice mice mice NM_001472541 Ighg 17.2 2.2 42 ± 30*  12 ± 8.7 NM_011314 Saa2 1.1 0.7 116 ± 45  27 ± 21 NM_011260Reg3g 0.9 0.9 1174 ± 959   11 ± 6.2 NM_054074 Defb6 1.3 0.6 47 ± 28 7.5± 4.6 NM_009312 Tac2 0.8 0.8 26 ± 10 6.9 ± 3.9 NM_001474025 Igh-VJ5581.0 1.0 22 ± 17 6.8 ± 2.6 NM_020268 Klk1b27 0.6 2.5 1.9 ± 0.2  10 ± 3.6*Expression fold value ± SD

FIG. 1 schematically shows that a low level (0.7 mGy/hr) of radiationsuppressed thymic cancer by activating immunity-related genes (Ighg,Saa2, Defb6, Reg3g, Tac2 and Igh-VJ558) and an apoptosis-related gene(Klk1b27) in thymocytes.

FIG. 2 is a graph showing the results obtained by irradiating AKR/J miceICR mice with a low level (0.7 mGy/hr) of radiation and measuring theweight of thymi of the mice at a time point (day 100) when the micestarted to die of thymic cancer during their housing, in order toanalyze the responses of genes sensitive to radiation based on thethymus weight. According to the present invention, genes that respondsensitively to a low level of radiation can be consistently measured byextracting thymi in an early stage of cancer development in which micestart to die of thymic cancer, and comparing the weights of theextracted thymi.

The invention claimed is:
 1. A method for detecting a gene sensitive toa radiation, the method comprising the steps of: irradiating acancer-induced AKR/J mouse and an ICR mouse with a gamma radiation at adose rate of 0.7 mGy/hr to a final dose of 1.7 Gy; extracting thymi fromthe AKR/J mouse and the ICR mouse; analyzing the thymi by a microarrayanalysis; selecting an apoptosis-related gene Klk1b27 from themicroarray analysis through a Venn diagram analysis; and amplifying thegene and measuring an expression level of the gene.
 2. The method ofclaim 1, wherein the cancer is thymic cancer.
 3. The method of claim 1,further comprising the step of extracting the thymi at a time point whenthe mouse starts to die of the cancer.
 4. The method of claim 1, whereinthe step of amplifying the Klk1b27 gene uses primers having sequencesset forth in SEQ ID NOS: 13 and 14.